1. Field of the Invention
The present invention relates to sensors for use in testing biological, biochemical, chemical or environmental samples, and methods of making and using the same.
2. Background of the Related Art
Analyses of biological, biochemical, chemical and environmental samples are invaluable, routinely used tools in health-related fields such as immunology, pharmacology, gene therapy, combinatorial chemistry, and the like. For example, in order to successfully implement therapeutic control of a biological process, it is imperative that a complete understanding of the binding between the species is obtained.
Many biochemical and biological analytical methods involve immobilization of a biological binding partner of a particular biological molecule on a surface, exposure of the surface and immobilized binding partner to a medium suspected of containing the biological molecule, and determination of the existence or extent of binding of the molecule to the surface-immobilized binding partner.
One such technique recently introduced involves surface plasmon resonance (SPR). Conventional SPR involves the use of a substrate, such as a glass slide, on one side of which is a thin metal film, a prism, a source of monochromatic and polarized light, a photodetector array and an analyte channel that directs a medium suspected of containing a particular analyte to the exposed surface of the metal film on the substrate. A face of the prism is separated from the second side of the substrate (i.e., the side opposite the metal film) by a thin film of refractive index matching fluid. Light from the light source is directed through the prism at an angle at which total internal reflection of the light results at the face of the prism. An evanescent field is generated as a result of this reflection, which extends from the prism into the metal film. This evanescent field can couple to an electromagnetic surface wave (a surface plasmon) at the metal film, causing surface plasmon resonance.
In such a device, coupling is achieved at a specific angle of incidence of the light with respect to the metal film (the SPR angle), at which the reflected light intensity goes through a minimum due to the resonance. This angle is determined by a photodetector array as the angle of reflectance and is highly sensitive to changes in the refractive index of a thin layer immediately adjacent to the surface of the metal film. Thus it is highly sensitive to coupling of an analyte to the metal film. For example, when a protein layer is adsorbed onto the metal surface from an analyte-containing medium delivered to the surface by the analyte channel, the SPR angle shifts to larger values and this shift is measured by the photodetector array. See, e.g., Stenberg et al., “Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabelled proteins,” Journal of Colloid and Interface Science, 43:2, 513–526 (1991); Homola et al., “Surface plasmon resonance sensors: review,” Sensors and Actuators, B 54, 3–15 (1999) and references cited therein. The instrumentation for analysis of biological samples using SPR is commercially available, for example, under the trade name BIAcore from Pharmacia Biosensor, Piscataway, N.J.
Although the introduction of SPR represented an extremely valuable contribution to the scientific community, current state-of-the-art commercial SPR instrumentation lacks the sensitivity needed to detect and analyze certain biological and chemical interactions that are at the vanguard of scientific research. Moreover, several complications have been observed with prior sensors for use in SPR which hinder the sensitivity of the technique.
For example, according to one technique for immobilizing a binding partner of an analyte on a surface plasmon resonance sensor, long chain hydroxy alkyl thiols are adsorbed onto a gold surface as a monolayer. The monolayer's exposed hydroxy groups are then activated with epichlorohydrin under basic conditions to form epoxides, which are then used to covalently attach a carboxylated dextran gel layer. A proteinaceous binding partner is then electrostatically adsorbed onto the dextran gel layer and subsequently covalently attached thereto. See, e.g., Lofas et al., “A novel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands,” J. Chem Soc., Chem. Comm., 1526–1528 (1990).
The effectiveness of this approach, however, is limited by several factors. First, covalent attachment of the proteinaceous binding partner to the dextran gel can affect the binding partners activity, or even viability. Second, covalent attachment of the binding partner to the gel generally cannot be effected with control over the orientation of the binding partner with respect to the surface of the sensor (and, more importantly, with respect to the analyte-containing medium to be tested). Third, non-specific interactions at the dextran gel are promoted by the negative charge that it carries.
According to another technique, a mixed monolayer of hydroxyl and biotin-terminated alkyl thiols is prepared on a gold surface and streptavidin is bound to the surface-bound biotin. Biotin-labeled proteins, which are the binding partners of the desired analytes, are then attached to the empty sites on the streptavidin. See, e.g., Spinke et al., “Molecular recognition at self-assembled monolayers: the construction of multicomponent multilayers,” Langmuir, 9, 1821–1825 (1993).
Because biotin must be covalently attached to the binding partner protein, however, this approach lacks control over orientation of the binding partner with respect to the analyte medium. Moreover, inactivation of the proteinaceous binding partner may also occur due to the formation of a covalent linkage.
Finally, as a practical limitation on its usefulness, conventional SPR reflectometry is difficult to realize in a large-scale array format because of the optics associated with the detection system. This is quite significant in view of the need for high-throughput biochemical assays based on protein arrays, such as those needed to measure the protein-protein and protein-ligand interactions for the many thousands of proteins identified by the human genome project as well as for DNA—DNA interactions in genomics.
One alternative to the use of conventional SPR methods involve colloidal surface plasmon resonance. Colloidal SPR is responsible for the intense colors exhibited by colloidal solutions of noble metals and is attributed to the collective oscillations of surface electrons induced by visible light. Colloidal SPR is an interfacial phenomenon, and can be used in two complementary modes to transduce binding events at the colloid surface.
In one mode, the optical signal arises from the dependence of the peak intensity and position of the surface plasmon absorbance of gold nanoparticles upon the local refractive index of the surrounding medium, which is altered due to binding at the colloid-solution interface. This mode, which is analogous to conventional SPR, has been previously utilized to determine biomolecular binding on the surface of a colloid in suspension. See, e.g., Englebienne, “Use of colloidal gold surface plasmon resonance peak shifts to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes,” Analyst, 123, 1599–1603 (1998); Eck et al., “Plasmon resonance measurements of the absorption and adsorption kinetics of a biopolymer onto gold nanocolloids,” Langmuir, 17, 957–960 (2001).
In the second mode, changes in the proximity of colloids due to their aggregation in suspension causes a large change in the absorbance spectrum of the colloidal suspension due to long-range coupling of surface plasmons. The interparticle distance-dependent color change of colloidal gold due to aggregation of gold colloids has been used in solution-based immunoassays and has more recently been employed to design a sensor capable of determining single base pair mismatches in DNA hybridization. See, e.g., Elghanian et al., “Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles,” Science, 277, 1078–1081 (1997); Storhoff et al., “One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticles probes,” J. AM. Chem. Soc., 120, 1959–1964 (1998); and PCT International Publication Number WO 01/51655.
Neither of these modes, however, is sufficient to remedy the disadvantages of conventional SPR methods. For example, much like conventional SPR, neither of these modes can be employed in a large-scale array format on a solid surface. Accordingly, there remains a need for a simple, SPR chip-based sensor for analyzing biological, biochemical, chemical and environmental samples.
The above references are incorporated by reference herein where appropriate for appropriate teachings of additional or alternative details, features and/or technical background.